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Clinical Infectious Diseases
EBV and Monoclonal Gammopathy of Clinical Significance in Autologous Stem CellTransplantation for Multiple Sclerosis
--Manuscript Draft--
Manuscript Number: CID-92283R2
Full Title: EBV and Monoclonal Gammopathy of Clinical Significance in Autologous Stem CellTransplantation for Multiple Sclerosis
Short Title: EBV complications in Auto-HSCT for MS
Article Type: Major Article
Corresponding Author: Varun Mehra, MRCP (UK), FRCPathKing's College HospitalLondon, UNITED KINGDOM
Corresponding Author SecondaryInformation:
Corresponding Author's Institution: King's College Hospital
Corresponding Author's SecondaryInstitution:
First Author: Varun Mehra, MRCP (UK), FRCPath
First Author Secondary Information:
Order of Authors: Varun Mehra, MRCP (UK), FRCPath
Elijah Rhone
Stefani Widya
Mark Zuckerman
Victoria Potter
Kavita Raj
Austin Kulasekararaj
Donal McLornan
Hugues de Lavallade
Nana Benson-Quarm
Christina Lim
Sarah Ware
Malur Sudhanva
Omar Malik
Richard Nicholas
Paolo A Muraro
Judith Marsh
Ghulam J Mufti
Eli Silber
Antonio Pagliuca
Majid A. Kazmi
Order of Authors Secondary Information:
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Manuscript Region of Origin: UNITED KINGDOM
Abstract: IntroductionAutologous haematopoietic stem cell transplantation (AHSCT) with anti-thymocyteglobulin (ATG) conditioning as treatment of active multiple sclerosis (MS) is rapidlyincreasing across Europe (EBMT registry data 2017). Clinically significant Epstein Barrvirus reactivation (EBV-R) following AHSCT with ATG for severe autoimmuneconditions is an under-recognised complication relative to T-cell deplete transplantsperformed for haematological diseases. This retrospective study reports EBV-Rassociated significant clinical sequelae in MS patients undergoing AHSCT with rabbitATG.MethodsRetrospective data was analysed for 36 consecutive MS-AHSCT patients at KingsCollege Hospital, London. All patients routinely underwent weekly EBV DNA PCRmonitoring and serum electrophoresis for monoclonal gammopathy (MG or M-protein).EBV-R with rising EBV viral load, M-protein and associated clinical sequelae werecaptured from clinical records.ResultsAll patients had evidence of rising EBV DNA-emia, including 7 who were lost to longterm follow-up, with a number of them developing high EBV viral load & associatedlymphoproliferative disorder (LPD). Nearly 72% (n-18/29) developed de-novo MG,some with significant neurological consequences with high M-protein and EBV-R. Sixpatients required anti-CD20 therapy (rituximab) with complete resolution of EBV relatedsymptoms. Receiver operating characteristics (ROC) estimated a peak EBV viraemiaof >500,000 DNA copies/ml correlated with high sensitivity (85.5%) & specificity(82.5%) (AUC-0.87; p-0.004) in predicting EBV-R related significant clinical events.ConclusionSymptomatic EBV reactivation increases risk of neurological sequelae and LPD in MS-AHSCT. We recommend regular monitoring for EBV and serum electrophoresis for MGbe mandated in MS patients in the first 3 months post AHSCT.
Response to Reviewers: ToDr Barbara D Alexander M.D.Associate EditorClinical Infectious Diseases
Dated: 30th Dec 2018
Dear Dr Alexander
Subject: Response to Reviewers
Manuscript Title:EBV and Monoclonal Gammopathy of Clinical Significance in Autologous Stem CellTransplantation for Multiple Sclerosis.
We would like to thank the journal for provisionally accepting our work. Considering thereviewer’s comments, we have made revisions to the manuscript with responsesoutlined for each of the queries raised by the reviewer, as below:
The Authors simply must have the manuscript edited for English grammar as many ofthe mistakes change the meaning of the sentence. Some (but not all) of the issues areas follows:Response:Please accept our apologies for the grammatical errors in the manuscript. We havereviewed and edited these errors where appropriate including the ones highlightedbelow.
Line 138 and line 357. deleted " be mandated". Cohort is too small to warrant"mandate"..but your data can lead to recommendation..Response:We have edited and replaced the word ‘mandate’ from the phrase.
Line 212-215. Please include the conversion factor for your assay to IU/ml in themethods section i.e 10 EBV DNA copies/ml=10 IU/ml
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Response:This has been rephrased within methods section; line 202-203
line 282: HAS versus IS?...I think "is"Response:Correction made to “is”
Line 298 and 300- not sure systemic sclerosis needs to be capitalized. But if so, needsto be so throughout manuscriptResponse:We have edited and removed un-necessary capitalisation for similar errors across themanuscript.
Lines 309-312. This sentence is not understandable based on current punctuation.Please address. ????This is further corroborated by the fact that similar LPD risk hasnot been observed in other ADs managed with ATG in our center. For example, amongpatients with Crohn' disease treated with ATG-AHSCT and those with severe aplasticanemia treated with ATG/cyclopsorin, only 52% (x/x) developed EBV-R (unpublisheddata) and none had LPD, suggesting that the problem may not be ATG specific.Response:Thank you for the suggestion. We have rephrased this to reflect our experience withother Autoimmune diseases (lines 310-315).
Line 319 delete the words "may still have"Response:correction made.
Line 330 seems to "be"?Response:correction made.
Line 340 "copies"/mlResponse:correction made.
Thank you again for your review of the revised manuscript. We hope these revisionsare satisfactory and will allow formal acceptance for publication.
Yours sincerely
On behalf of all co-authors:
Dr Varun Mehra, MRCP(UK), FRCPath Dr Majid Kazmi; FRCP(UK) FRCPathDepartment of Haematological Medicine Chief of Cancer Division & ConsultantHaematologistKings College Hospital, London, UK Kings, Guy’s & St Thomas’ Hospital, London,[email protected] ; [email protected]; PH-004478027617716
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To Professor Robert Schooley, M.D. The Editor-in-Chief Clinical Infectious Diseases Dated: 20th Dec 2018 Dear Professor Schooley (Editor-in-Chief) and Dr Alexander (Associate Editor) We are pleased to submit our revised article entitled; “EBV & Monoclonal Gammopathy of Clinical Significance in Autologous Stem Cell Transplantation for Multiple Sclerosis” for consideration of publication in your internationally reputed journal, Clinical Infectious Diseases. Just to summarise again: Autologous Stem Cell Transplants (AHSCT) with anti-thymocyte globulin ATG) based conditioning is a novel approach to treatment of active multiple sclerosis (MS) and recent data from MIST study collaborators (Burt et al; Clinical Trial Registry: NCT00273364) have shown some exciting preliminary results showing superiority of AHSCT over established disease modifying therapies, confirming results from other UK and international studies in this field. However, as the evidence builds, safety aspects of these procedures needs to be seriously considered. This study reports rates of Epstein Barr virus (EBV) reactivation and associated clinical sequelae with monoclonal gammopathy (M-protein), in cohort of Multiple Sclerosis patients who underwent ATG conditioned immunosuppressive AHSCT in a single centre. We report a significantly higher proportion of MS patients had detectable EBV DNA post-AHSCT; were more likely to develop clinically significant EBV viraemia of >500,000 DNA copies/ml and develop de-novo M-protein of clinical significance with clinical events ranging from probable lymphoproliferative disorders and disabling neurological complications, unrelated to MS. This report of significant clinical complications related to EBV and M-protein, possibly reflect underlying altered immunopathological state of MS disease and its interactions with reactivation of EBV virus, which if monitored and treated pre-emptively may reduce associated morbidity and improve outcomes. To help readers, we have also described two interesting clinical vignettes as a supplementary to this report, highlighting significant risk of neurological events following development of M-protein, triggered following EBV reactivations in MS patients. We can confirm that this manuscript has not been published and is not under consideration for publication elsewhere. All authors have seen, approved and contributed to this work. We have no conflicts of interest to disclose. We believe that this report fits well within the scope of your journal, highlighting important clinical message about EBV complications in ATG conditioned AHSCT for MS and will appeal to journal’s readers interested in infectious complications related to immunosuppressive therapies including AHSCTs for autoimmune conditions, with a potential to change clinical practice in this area. We have provided point to point responses to the reviewer’s comments. Thank you for your consideration of this revised manuscript and looking forward to your acceptance. Yours Sincerely On behalf of all co-authors: Dr Varun Mehra, MRCP(UK), FRCPath Dr Majid Kazmi; FRCP(UK) FRCPath Department of Haematological Medicine Chief of Cancer Division & Consultant Haematologist Kings College Hospital, London, UK Kings, Guy’s & St Thomas’ Hospital, London, UK [email protected] ; Ph-004478865087013 [email protected]; PH-004478027617716
Cover Letter Click here to access/download;Cover Letter;EBV MS CIDEditor revised Cover Letter.docx
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EBV and Monoclonal Gammopathy of Clinical Significance in Autologous 1
Stem Cell Transplantation for Multiple Sclerosis. 2
Running Title: EBV complications in Auto-HSCT for MS 3
Varun Mehra*, Elijah Rhone*, Stefani Widya, Mark Zuckerman, Victoria Potter, Kavita Raj, 4
Austin Kulasekararaj, Donal McLornan, Hugues de Lavallade, Nana Benson-Quarm, Christina 5
Lim, Sarah Ware, Malur Sudhanva, Omar Malik, Richard Nicholas, Paolo A Muraro, Judith 6
Marsh, Ghulam J Mufti, Eli Silber, Antonio Pagliuca and Majid A. Kazmi 7
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*These authors contributed equally to this work as 1st Authors. 9
10
Key Points: 11
EBV reactivation is common post-transplant with ATG for multiple sclerosis (MS), 12
with significant lymphoproliferative & neurological sequelae associated with rising 13
M-protein. Serial monitoring of EBV & M-protein is recommended post-transplant, as 14
is pre-emptive anti-CD20 therapy with EBV DNA >500,000 copies/ml. 15
16
Corresponding authors: 17
1. Dr Varun Mehra; [email protected]; +442032995378 18
Alternate Corresponding Author: 19
2. Dr Majid Kazmi; [email protected] ; +442071882757 20
21
Key Words: 22
Multiple Sclerosis; Autologous Hematopoietic Stem Cell Transplantation, Epstein-23
Barr Virus Infection; Monoclonal Gammopathy; Post-transplant Lymphoproliferative 24
Disorder 25
26
Full Manuscript (clean copy) in .doc format only [no PDFs] Click here to access/download;Full Manuscript in .doc formatonly [no PDFs];EBV in MS updated CID revision-clean copy
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Author Affiliations: 27
1. Dr Varun Mehra*; Department of Haematology, King’s College Hospital NHS Foundation 28 Trust, Denmark Hill, London, United Kingdom. 29
2. Dr Elijah Rhone*: Department of Neurology, King’s College Hospital NHS Foundation Trust, 30 Denmark Hill, London, United Kingdom 31
3. Stefani Widya: GKT School of Medical Education, Kings College London University, 32 London 33
4. Dr Mark Zuckerman: Department of Virology, King’s College Hospital NHS Foundation 34 Trust, Denmark Hill, London, United Kingdom. 35
5. Dr Victoria Potter: Department of Haematology, King’s College Hospital NHS Foundation 36 Trust, Denmark Hill, London, United Kingdom. 37
6. Dr Kavita Raj: Department of Haematology, King’s College Hospital NHS Foundation Trust, 38 Denmark Hill, London, United Kingdom AND Department of Haematology, Guy’s and St. 39 Thomas’ NHS Foundation Trust, London, United Kingdom 40
7. Dr Austin Kulasekararaj: Department of Haematology, King’s College Hospital NHS 41 Foundation Trust, Denmark Hill, London, United Kingdom. 42
8. Dr Donal McLornan: Department of Haematology, King’s College Hospital NHS Foundation 43 Trust, Denmark Hill, London, United Kingdom AND Department of Haematology, Guy’s and 44 St. Thomas’ NHS Foundation Trust, London, United Kingdom 45
9. Dr Hugues de Lavallade: Department of Haematology, King’s College Hospital NHS 46 Foundation Trust, Denmark Hill, London, United Kingdom. 47
10. Nana Benson-Quarm: Department of Haematology, King’s College Hospital NHS 48 Foundation Trust, Denmark Hill, London, United Kingdom. 49
11. Christina Lim: Department of Haematology, King’s College Hospital NHS Foundation Trust, 50 Denmark Hill, London, United Kingdom. 51
12. Sarah Ware: Department of Haematology, King’s College Hospital NHS Foundation Trust, 52 Denmark Hill, London, United Kingdom. 53
13. Dr Malur Sudhanva: Department of Virology, King’s College Hospital NHS Foundation 54 Trust, Denmark Hill, London, United Kingdom 55
14. Dr Omar Malik: Department of Neurology, Imperial College Healthcare, London, United 56 Kingdom 57
15. Dr Richard Nicholas: Department of Neurology, Imperial College Healthcare, London, 58 United Kingdom 59
16. Professor Paolo A. Muraro: Department of Neurology, Imperial College Healthcare, 60
London, United Kingdom AND Department of Neuroimmunology, Imperial College London, 61 London, United Kingdom 62
17. Professor Judith Marsh: Department of Haematology, King’s College Hospital NHS 63
Foundation Trust, Denmark Hill, London, United Kingdom 64 18. Professor
Ghulam J. Mufti: Department of Haematology, King’s College Hospital NHS 65
Foundation Trust, Denmark Hill, London, United Kingdom 66 19. Dr Eli Silber: Department of Neurology, King’s College Hospital NHS Foundation Trust, 67
Denmark Hill, London, United Kingdom 68 20. Professor
Antonio Pagliuca: Department of Haematology, King’s College Hospital NHS 69
Foundation Trust, Denmark Hill, London, United Kingdom 70 21. Dr Majid Kazmi: Department of Haematology, King’s College Hospital NHS Foundation 71
Trust, Denmark Hill, London, United Kingdom AND Department of Haematology, Guy’s and 72 St. Thomas’ NHS Foundation Trust, London, United Kingdom 73
74
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Conflict of interest: The authors declare no competing financial interests as below: 78
1. Varun Mehra- no competing financial interests 79
2. Elijah Rhone- no competing financial interests 80
3. Stefani Widya- no competing financial interests 81
4. Mark Zuckerman- no competing financial interests 82
5. Victoria Potter- no competing financial interests 83
6. Kavita Raj- no competing financial interests 84
7. Austin Kulasekararaj- no competing financial interests 85
8. Donal McLornan- no competing financial interests 86
9. Hugues de Lavallade- no competing financial interests 87
10. Nana Benson-Quarm- no competing financial interests 88
11. Christina Lim- no competing financial interests 89
12. Sarah Ware- no competing financial interests 90
13. Malur Sudhanva- no competing financial interests 91
14. Omar Malik- no competing financial interests 92
15. Richard Nicholas- no competing financial interests 93
16. Paolo A Muraro- no competing financial interests 94
17. Judith Marsh- no competing financial interests 95
18. Ghulam J Mufti- no competing financial interests 96
19. Eli Silber- no competing financial interests 97
20. Antonio Pagliuca- no competing financial interests 98
21. Majid A. Kazmi- no competing financial interests 99
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Abstract 111
Introduction 112
Autologous haematopoietic stem cell transplantation (AHSCT) with anti-thymocyte globulin 113
(ATG) conditioning as treatment of active multiple sclerosis (MS) is rapidly increasing across 114
Europe (EBMT registry data 2017). Clinically significant Epstein Barr virus reactivation (EBV-115
R) following AHSCT with ATG for severe autoimmune conditions is an under-recognised 116
complication relative to T-cell deplete transplants performed for haematological diseases. 117
This retrospective study reports EBV-R associated significant clinical sequelae in MS patients 118
undergoing AHSCT with rabbit ATG. 119
Methods 120
Retrospective data was analysed for 36 consecutive MS-AHSCT patients at Kings College 121
Hospital, London. All patients routinely underwent weekly EBV DNA PCR monitoring and 122
serum electrophoresis for monoclonal gammopathy (MG or M-protein). EBV-R with rising 123
EBV viral load, M-protein and associated clinical sequelae were captured from clinical 124
records. 125
Results 126
All patients had evidence of rising EBV DNA-emia, including 7 who were lost to long term 127
follow-up, with a number of them developing high EBV viral load & associated 128
lymphoproliferative disorder (LPD). Nearly 72% (n-18/29) developed de-novo MG, some with 129
significant neurological consequences with high M-protein and EBV-R. Six patients required 130
anti-CD20 therapy (rituximab) with complete resolution of EBV related symptoms. Receiver 131
operating characteristics (ROC) estimated a peak EBV viraemia of >500,000 DNA copies/ml 132
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correlated with high sensitivity (85.5%) & specificity (82.5%) (AUC-0.87; p-0.004) in 133
predicting EBV-R related significant clinical events. 134
Conclusion 135
Symptomatic EBV reactivation increases risk of neurological sequelae and LPD in MS-136
AHSCT. We recommend regular monitoring for EBV and serum electrophoresis for MG in 137
MS patients in the first 3 months post AHSCT 138
139
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INTRODUCTION: 151
Multiple sclerosis (MS) is a chronic autoimmune, inflammatory, demyelinating disease of the 152
central nervous system[1]-[2], with a relapsing-remitting (RRMS) presentation in the majority 153
of patients at diagnosis. Recovery from relapses may be complete or partial[3],[4]. After a 154
variable period of time, people with RRMS may develop a more progressive disability 155
accumulation with or without superimposed relapses; termed secondary progressive multiple 156
sclerosis (SPMS). A minority experience progressive disability from the onset of disease, 157
termed primary progressive multiple sclerosis (PPMS)[4]. A number of immunomodulatory 158
disease modifying therapies (DMTs) are currently licensed for treatment of RRMS with an 159
aim of reducing number of relapses and accrual of disability, although with variable 160
efficacy[5]. Since 1996, Autologous Hematopoietic Stem Cell Transplantation (AHSCT) has 161
been a novel approach for MS management, using immunoablation followed by 162
immunomodulation mechanisms, with evidence of significant suppression of inflammatory 163
activity and qualitative changes in the reconstituted immune system (immune reset theory)[6–164
8]. AHSCT appears most effective for MS patients with evidence of inflammatory activity on 165
MRI, younger age, a shorter disease duration, low to moderate disability levels (Expanded 166
Disability Status Scale [EDSS] <6 or up to 6.5 if recent progression) and failure of at least 1 167
highly active DMT (natalizumab or alemtuzumab) with no significant comorbidities[9–11]. 168
Recently reported preliminary results of randomised MIST study[12] found AHSCT to be 169
superior to standard disease modifying therapy (DMT) for RRMS with respect to both 170
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treatment failure and disability progression. 171
172
However, risk of subsequent rise in opportunistic infections following such 173
immunosuppressive therapies remain a potential concern[13]. MS patients undergoing 174
AHSCT have often been exposed to a number of immunomodulating DMTs; the addition of 175
immunosuppressive rabbit anti-thymocyte globulin (rATG) to their conditioning regimen may 176
confer a higher risk of viral reactivation in these patients. The number of AHSCTs performed 177
for MS is rising significantly in Europe[14] and as more centres perform AHSCT for this 178
indication, it is increasingly important to recognise the unique problems faced by these 179
patients post AHSCT. This retrospective study reports for the first time, EBV-R associated 180
neurological sequelae and lymphoproliferative disorder (LPD) in MS patients undergoing 181
rATG conditioned AHSCT in our centre. 182
183
METHODS 184
Patients and procedures 185
Data was collected retrospectively on 36 consecutive MS patients undergoing AHSCT 186
between February 2012 and February 2017 at Kings College Hospital, London. Peripheral 187
blood stem cells were collected following standard mobilisation strategy consisting of 188
cyclophosphamide 4g/m2 over 2 days and granulocyte colony-stimulating factor for 7 days. 189
Thirty-five (97%) patients were conditioned using standard protocol of cyclophosphamide 190
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(50mg/kg for 4 days) and rATG (2.5mg/kg/day for 3 days) for in-vivo lymphodepletion 191
followed by stem cell infusion. One patient was conditioned with 192
carmustine/etoposide/cytarabine/melphalan regimen along with an equivalent dose of rATG 193
(BEAM-ATG) prior to stem cell infusion. The median CD34 stem cell dose returned was 7.17 194
x10^6/kg (range 4.0-17.1x10^6/kg). 195
196
Prior exposure to EBV was assessed by serological evidence (EBV viral capsid antigen; VCA 197
IgG). EBV DNA load monitoring was performed on whole blood samples by standardised 198
quantitative real-time polymerase chain reaction (RT-PCR) using Rotor-GeneTM (Qiagen) 199
assay of EBV BZLF1 DNA. This assay was adapted from our published assay using 200
LightCycler (Roche)[15] and since been validated against the recently published WHO 201
standard, with our lab’s EBV DNA quantification of 10 copies/ml considered equivalent to 10 202
IU/ml DNA reported with the WHO reference method[16]. EBV-R was defined as rising EBV 203
DNA load of >10 copies/millilitre (ml) detected on two consecutive tests based on our assay 204
sensitivity. Symptomatic EBV-R was captured by peak blood EBV DNA load, presence of B 205
symptoms (defined by presence of either unexplained weight loss, recurrent fever, night 206
sweats); which was in-turn defined by clinical, radiological and/or histological evidence based 207
on recent ECIL-6 guidelines[17]. In addition, significant ‘clinical events’ were also defined as 208
new & persistent organ dysfunction (e.g. neurological events) temporally associated with 209
rising EBV viraemia in MS patients. Serum protein electrophoresis was routinely tested 210
around 3 months post HSCT as part of our institutional practice, with immunoglobulin 211
subclasses identified by immunofixation electrophoresis. Patient outcomes were assessed at 212
last follow up as of April 2017. 213
214
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Statistics 215
The database of transplants and outcomes was built in Microsoft Excel 2016 and statistical 216
analyses were performed using IBM SPSS Statistics version 24.0. Patient characteristics are 217
presented as medians (with inter-quartile ranges; IQR) for data with non-normal distribution. 218
Comparisons of baseline characteristics used Mann-Whitney U test, Fisher’s exact test, or 219
Chi-squared test for trend as appropriate. Receiver Operating characteristics (ROC) curve 220
was obtained correlating LPD and clonal gammopathy associated clinical events with rising 221
EBV viraemia (copies/ml). 222
223
RESULTS 224
Baseline characteristics are presented in Table 1. Most MS patients (88.9%) had RRMS 225
phenotype with median of 2 (range 0-3) DMTs prior to AHSCT. Twenty-two MS patients had 226
prior exposure to natalizumab and 7 were treated with Alemtuzumab (6 patients received 227
both). All 36 (100%) MS patients were serologically positive for EBV VCA IgG pre-treatment, 228
indicating prior EBV exposure and had detectable EBV DNA post-AHSCT. Seven MS 229
patients were lost to long-term follow up for EBV monitoring. The median time to first EBV 230
DNA detection post-transplant was 30 days (IQR 23-46 days). EBV DNA levels peaked at a 231
median of 32 days post-transplant (IQR 31-53 days). All MS patients had normal baseline 232
lymphocyte counts (median) pre-HSCT with a median time of 46 days (range 14-404 days) 233
to lymphocyte recovery (defined by total lymphocyte count >1.0x106/ml) following AHSCT 234
(See Figure 1). A high proportion (86%; n-25/29) of the MS patients in active follow-up 235
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recovered lymphocyte counts around D56 with a median lymphocyte count of 1.56 (106 236
cells/ml); Four patients remained lymphopenic at last follow up. 237
238
All patients were stratified into following 3 groups according to peak rise/burden of EBV DNA-239
aemia (copies/ml): <100,000 (<100k) copies/ml, 100,001-500,00 (100k-500k) copies/ml and 240
>500,000 (>500k) copies/ml to identify any specific thresholds for clinically significant events 241
related to rising EBV-R (Table 1). The majority of patients (76%) with rising EBV viral load 242
>100k copies/ml were routinely screened by computed tomographic (CT) scans to assess for 243
evidence of LPD, in line with our institutional policy. One third (34.5%) of patients developed 244
peak EBV viraemia of >500k copies/ml. Eight patients (27.6%) developed symptomatic EBV-245
R; defined as persistent fever, lymphadenopathy and/or B symptoms. Of these 8 patients, 246
only 1 (12.5%) had a peak EBV viraemia <100kDNA copies/ml with the remaining 7 (87.5%) 247
patients having a peak EBV viraemia of >500k copies/ml. Three patients with rising EBV 248
viraemia >500k copies/ml had findings consistent with probable LPD on CT imaging; 249
however, none had definitive histological diagnosis. Three MS patients had worsening 250
neurological symptoms concurrent with rising EBV viraemia >500k copies/ml and clonal 251
gammopathy, as described below. 252
253
Interestingly, we also observed frequent de novo monoclonal gammopathy (MG or M-protein) 254
in 18 MS patients (62.4%) following rising EBV viraemia; the majority (n-16) of whom 255
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developed IgG subtype and the remaining 2 developed IgA and IgM M-protein. Concerningly 256
two of these patients developed clinically significant M-Protein burden; one patient with IgG 257
Kappa M-protein of 45.6g/L developed hyper-viscosity and neurological symptoms mimicking 258
MS relapse, requiring plasma exchange. Another patient developed significant lumbosacral 259
radiculopathy with rising EBV-R and high IgM lambda M-protein (IgM 48.5g/L) (see 260
supplementary case vignettes). Figure 2 highlights the association of neurological 261
symptom onset following rising EBV viraemia (log copies), falling lymphocyte counts (x 262
106/ml) with significant rise in M-protein (gm/lt) levels post AHSCT. A third patient developed 263
painful lower limb paraesthesia following rising EBV viraemia >500k copies/ml, although did 264
not have any M-protein detected. Their symptoms persisted at last follow up despite no 265
evidence of MS related new disease activity. 266
267
Six patients were treated with anti-CD20 antibody, rituximab (375mg/m2 weekly up to 4 268
weeks), due to clinical severity of EBV reactivations and leading to reduction in EBV viral 269
load and concurrent improvement/resolution of EBV related symptoms. ROC curve analysis 270
(Figure 3) confirmed EBV viraemia of >500k copies/ml correlated with high sensitivity 271
(85.5%) and specificity (82.5%) (AUC-0.87; 95%CI-0.73-1.0; p-0.004) in predicting significant 272
EBV related clinical events (evidence of LPD and/or neurological symptoms) that may require 273
treatment with rituximab. The sensitivity dropped significantly on lower estimates for events 274
below 500k copies/ml. 275
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276
The median time to resolution of EBV viraemia post-rituximab was 21 days (IQR 19-124 days) 277
in 5 patients with >500k copies/ml (one patient was treated for late onset persistent 278
symptomatic clonal gammopathy, despite fall in EBV levels). No significant adverse events 279
were noted in the treated group. Nine patients had a persistent low level EBV viraemia 280
detectable at last follow up. All patients who underwent AHSCT were alive as of April 2017. 281
282
DISCUSSION: 283
MS as an autoimmune disorder (AD) is theorised to have generally similar underlying 284
pathophysiological immune dysregulation mechanisms[18–22] relative to other chronic 285
autoimmune conditions. Epstein-Barr virus (EBV) is increasingly implicated in the 286
pathogenesis of MS by virtue of epidemiological and serological evidence, impaired CD8+ T 287
cell immune responses to EBV and possible underlying genetic susceptibility for 288
autoimmunity (with EBV encoded protein interactions), as recently described by Harley et al 289
and others[1,23–25]. 290
291
Generally, EBV-R related LPD has been reported in both allogeneic (allo-HSCT) and solid 292
organ transplants treated with immunosuppressive therapy, often with a significant impact 293
on organ function and overall survival[26–30]. It is observed that reduced intensity allo-HSCT 294
for malignant haematological conditions using alemtuzumab have a relatively lower overall 295
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risk of LPD compared to ATG based treatments, possibly mediated by more effective pan-B 296
& T cell lymphodepletion. In contrast, ATG primarily affects the T-cell repertoire with delayed 297
EBV specific CD8+ T cell recovery[31]. Clinically significant endogenous viral infections 298
including EBV following ATG conditioned AHSCT for severe ADs such as Crohn’s disease 299
and systemic sclerosis is increasingly recognised, but the development of lymphoproliferative 300
disorders (LPD) remains rare in these ADs[13,32,33]. Nash et al[32] concerningly reported 301
2 deaths (1 MS and 1 systemic sclerosis patient) from EBV related LPD in 56 ATG 302
conditioned AHSCT for autoimmune diseases. Additionally, EBV associated 303
haemophagocytosis in ATG-AHSCT for ADs have also been reported[34], with one resulting 304
in death of the patient[35]. 305
306
Our report of suspected EBV related LPDs (~10%) in MS-AHSCT group is relatively higher 307
than published reports with allo-HSCTs (4.5-7%)[36,37] and our own centre’s unpublished T-308
cell depleted allo-HSCT experience (6.5%); possibly a reflection of underlying 309
immunopathological state of MS itself[38]. This is further corroborated by the fact that similar 310
LPD risk has not been observed in other ADs, e.g. Crohn’s disease, treated with ATG -311
AHSCT in this centre. Another example from our centre’s experience of severe aplastic 312
anaemia (n-40) treated with ATG/ciclosporin, only 52% (n-21/40) developed EBV-R 313
(unpublished data) and none had LPD or required any treatment, suggesting that the problem 314
may not be ATG specific. 315
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316
Our study’s observation of significant persistent neurological events (with no evidence of new 317
MS disease activity) associated with clonal gammopathy suggest a potentially new clinical 318
syndrome, described for the first time in ATG conditioned AHSCTs in MS and possibly 319
induced by clonal B cell dysregulation following EBV-R. It could be hypothesised that any 320
remaining EBV infected latent B cells, surviving despite high doses of cyclophosphamide 321
(given with mobilisation and conditioning in MS ASHCT)[8] and compounded by depletion of 322
CD8+ T cells by ATG, may serve as potential source for EBV escape while interacting 323
abnormally within the host immune micro-environment[39] and leading to rise in M-protein, 324
LPD and neuro-inflammatory insults in some of the MS patients post AHSCT. Reports of 325
lower incidence of EBV-R and LPD in another commonly used protocol for MS-AHSCT using 326
BEAM-ATG (Ricardo Saccardi, Carregi University Hospital, Florence; personal 327
communication) may reflect the greater myeloablative effect of BEAM chemotherapy which 328
could further deplete the residual B cell pool and thus lower potential for EBV proliferation. It 329
is plausible that dose of rATG is critical, given we have not seen similar reports from other 330
centres where less rATG doses were given for MS-AHSCT (range between 5.0-6.5 mg/Kg; 331
personal communication) but there seems to be some variability in prospective serial EBV 332
monitoring in these patients. 333
334
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The clinical threshold for EBV viral load as a significant risk factor for post-transplant LPD is 335
widely debated. This study reports peak EBV viral load >500k copies/ml post AHSCT is 336
significantly associated with probable LPD and neurological events in MS patients with high 337
sensitivity (85.5%) and specificity (82.5%) (p-0.004) (Fig 3, ROC curve). Our ROC curve 338
estimates are potentially limited by the relatively small number of events analysed but this 339
has consistently been useful in our MS-AHSCT experience for predicting clinical events with 340
high EBV load. Our EBV PCR assay has been validated against the recently defined standard 341
WHO reference method (i.e. 10 copies/ml=10 IU/ml EBV DNA) [16] and thus this EBV 342
threshold for pre-emptive treatment with Rituximab, can potentially be applied in relevant 343
clinical context in other centres using similar validated essays. Rituximab treatment delivered 344
good overall response in our symptomatic patients, with resolution of EBV related clinical 345
symptoms and no subsequent viral or bacterial infections at last follow up. The role of 346
prophylactic or pre-transplant rituximab in MS-AHSCT is also a potential area of interest in 347
reducing risk of EBV-LPD in stem cell transplants as observed by Burns et al[36] and future 348
randomised studies are required to investigate its potential benefit. 349
350
Our study limitations include its retrospective nature and that no suspected LPD patients had 351
histological confirmation, mainly related to patient refusal or technical difficulties. Seven MS 352
patients were lost to follow up for EBV monitoring following discharge, which limits the 353
- 16 -
findings of this study. Additionally, our numbers were too small to identify any association of 354
EBV related clinical events with previous DMT exposure in MS patients. 355
356
In conclusion, symptomatic EBV reactivation increases risk of neurological sequelae and 357
LPD in MS-AHSCT. Regular monitoring for rising EBV viraemia, as recommended by 358
Snowden et al [10] and Muraro et al [11], and serum electrophoresis for M-protein should be 359
considered in the first 3 months post-AHSCT for MS. We recommend persistent high EBV 360
viraemia > 500k DNA copies/ml as potential trigger for consideration of pre-emptive anti-361
CD20 therapy and potentially reduce associated morbidity. 362
363
364
365
366
367
368
Acknowledgements: To our patients and their families and carers in supporting this study. 369
370
371
372
373
374
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376
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510
511
512
513
514
515
516
517
518
519
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521
522
523
524
525
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Table 1: Baseline patient characteristics and EBV related clinical events according to peak 526
EBV DNA-aemia burden. 527
528
Baseline characteristics (n-36)
Patient Groups according to peak
EBV DNA in copies/ml
(n-29)
0 - 100,000 100,001
- 500,000
>500,000
Median age at time of AHSCT in years (range)
43.5 (36– 47)
No of patients (%) 16 (55.2) 3 (10.3) 10 (34.5)
Gender Male Female
19 (52.8%) 17 (47.2%)
M-Protein (n) 11 0 7
Disease Type (n; %) Relapsing Remitting MS Secondary Progressive MS Primary Progressive MS
22 (61.1%) 10 (27.8%) 4 (11.1%)
Median EBV DNA log value at peak
(IQR)
4.8 (3.5-4.8)
5.5 (N/A)
6.25 (6.1-6.9)
Median number of previous DMT (range)
2 (0 – 6) Median number of
prior DMTs 2 3 2
Previous use of high efficacy DMT (n) Natalizumab Alemtuzumab Both
22 8 6
Symptomatic EBV (n)
1 0 7
Median EDSS (range) 6.0
(2.5 – 8.0) LPD diagnosis (CT/Biopsy) (n)
0 0 3
by CT alone
Median follow up post AHSCT in days (range)
436 (188 – 785)
Neuro/autoimmune complications (n)
0 0 3
No. of patients with prior EBV exposure (n; %)
36 (100%) Treated with Rituximab (n)
0 0 6
No. of patients with detectable EBV post AHSCT (n; %)
29 (80.5%) Confirmed EBV resolution at last
follow up (n) 7 2 7
No. of patients lost to long term follow up (n; %)
7 (19.5%) Detectable EBV DNA at last follow up (n)
9 1 3
Median Time to EBV detection post AHSCT in days (IQR)
30 (23-46) Median time for EBV
resolution (IQR in days)
67 days (44-155)
47 days (N/A)
63 days (45 - 170)
Median Time to peak EBV DNA levels in days (IQR)
32 (31-53) Median Time to peak EBV DNA levels in
days (IQR)
40 days (25-85)
30 days (N/A)
39 days (32-43)
529
Abbreviations: 530
AHSCT- Autologous Haematopoietic Stem cell transplant; CT- computed tomography; DMT- 531
Disease modifying therapy; EBV- Epstein Barr virus; EDSS- Kurtzke Expanded Disability 532
Status Scale; IQR- Interquartile range; LPD- Lymphoproliferative disease; M-Protein: 533
Monoclonal paraprotein or gammopathy; MS- Multiple Sclerosis 534
- 22 -
Figure Legends 535
536
Figure 1: Trend of Lymphocyte count recovery following ATG in MS patients. 537
538
Legend: This figure shows trends of lymphocyte count from baseline to recovery post 539
AHSCT for MS patients. Majority of patients had normal baseline lymphocyte counts pre-540
HSCT and became lymphopenic post ATG with counts recovering towards d+28 and >85% 541
of patients recovered counts by d+56, with some overshooting from their baseline, possibly 542
reflective of EBV related lymphoproliferation in some of these patients. 543
544
AHSCT: Autologous Haematopoietic Stem Cell Transplantation; ATG: Anti-Thymocyte 545
Globulin; d+: Days post AHSCT; MS: Multiple Sclerosis. 546
547
548
Figure 2: Paraprotein, EBV & Lymphocyte trends in two MS patients with significant 549
neurological sequelae post AHSCT. 550
551
Legend: This figure demonstrates trends of EBV copies (log), paraprotein levels (g/lt) and 552
Lymphocyte levels (counts x10^6/ml) in two MS patients with significant neurological 553
symptoms following EBV reactivation. Both patients had significant EBV viraemia (log>5.2 554
or >500,000 copy number) and developed significant paraproteinaemia, which was only 555
noted after persistent unexplained neurological symptoms. The trend reversed following 556
anti-CD20 (rituximab) therapy, with limited recovery in neurological symptoms. 557
558
D: Days post AHSCT; EBV-R: Epstein Barr Virus reactivation; MS: Multiple sclerosis; 559
AHSCT: Autologous Haematopoietic Stem Cell Transplants. 560
561
562
Figure 3: ROC curve estimates for peak EBV viraemia levels and significant clinical 563
events in MS post AHSCT. 564
565
Legend: ROC curve demonstrating significant correlation between high EBV levels and 566
clinical events (LPD & neurological events) in MS-AHSCT patients, with highest sensitivity 567
and specificity noted with peak EBV viraemia of >500,000 copies/ml (p-0.0004). 568
569
EBV: Epstein Barr Virus; LPD- Lymphoproliferative disorder; MS-AHSCT: Multiple Sclerosis 570
patients treated with autologous haematopoietic stem cell transplants; ROC: receiver 571
operating characteristics 572
573 574 575 576 577 578 579
580
- 23 -
581
Figure 1: Trend of Lymphocyte count recovery following ATG conditioned AHSCT in 582
MS patients. 583
584
585 586
587
Abbreviations: 588
AHSCT: Autologous Haematopoietic Stem Cell Transplantation; ATG: Anti-Thymocyte 589
Globulin; d+: Days post AHSCT; MS: Multiple Sclerosis. 590
591
592
593
594
595
596
597
598
Lymphocyte count trends post ATG in MS-AHSCT
Time (in days) since ATG in MS-AHSCT
- 24 -
Figure 2. Paraprotein, EBV & Lymphocyte trends in two MS patients with significant 599
neurological sequelae post AHSCT 600
601
602 603 604 605 606 607
608
Abbreviations: 609
D: Days post AHSCT; EBV-R: Epstein Barr Virus reactivation; MS: Multiple sclerosis; 610
AHSCT: Autologous Haematopoietic Stem Cell Transplants. 611
612
613
614
615
616
617
618
619
620
- 25 -
621
Figure 3: ROC curve estimates for peak EBV viraemia levels and significant clinical 622
events in MS post AHSCT. 623
624
625
626
Abbreviations: 627
628
EBV: Epstein Barr Virus; LPD- Lymphoproliferative disorder; MS-AHSCT: Multiple Sclerosis 629
patients treated with autologous haematopoietic stem cell transplants; ROC: receiver 630
operating characteristics 631
632
633
634
635
636
637
638
639
EBV DNA in
copies/ml
Sensitivity Specificity
0-50,000 100% 50%
50,001-
100,000
85.5% 60.1%
100,001-
500,000
85.5% 73.7%
>500,001 85.5% 82%
>700,000 71.4% 82%
AUC-0.87 (95% CI: 0.73-1.0) AUC-0.87 (95% CI: 0.73-1.0); p-0.004
Table 1: Baseline patient characteristics and EBV related clinical events according to
peak EBV DNA-aemia burden.
Baseline characteristics (n-36)
Patient Groups according to peak
EBV DNA in copies/ml
(n-29)
0 - 100,000 100,001
- 500,000
>500,000
Median age at time of AHSCT in years (range)
43.5 (36– 47)
No of patients (%) 16 (55.2) 3 (10.3) 10 (34.5)
Gender Male Female
19 (52.8%) 17 (47.2%)
M-Protein (n) 11 0 7
Disease Type (n; %) Relapsing Remitting MS Secondary Progressive MS Primary Progressive MS
22 (61.1%) 10 (27.8%) 4 (11.1%)
Median EBV DNA log value at peak
(IQR)
4.8 (3.5-4.8)
5.5 (N/A)
6.25 (6.1-6.9)
Median number of previous DMT (range)
2 (0 – 6) Median number of
prior DMTs 2 3 2
Previous use of high efficacy DMT (n) Natalizumab Alemtuzumab Both
22 8 6
Symptomatic EBV (n)
1 0 7
Median EDSS (range) 6.0
(2.5 – 8.0) LPD diagnosis (CT/Biopsy) (n)
0 0 3
by CT alone
Median follow up post AHSCT in days (range)
436 (188 – 785)
Neuro/autoimmune complications (n)
0 0 3
No. of patients with prior EBV exposure (n; %)
36 (100%) Treated with Rituximab (n)
0 0 6
No. of patients with detectable EBV post AHSCT (n; %)
29 (80.5%) Confirmed EBV resolution at last
follow up (n) 7 2 7
No. of patients lost to long term follow up (n; %)
7 (19.5%) Detectable EBV DNA at last follow up (n)
9 1 3
Median Time to EBV detection post AHSCT in days (IQR)
30 (23-46) Median time for EBV
resolution (IQR in days)
67 days (44-155)
47 days (N/A)
63 days (45 - 170)
Median Time to peak EBV DNA levels in days (IQR)
32 (31-53) Median Time to peak EBV DNA levels in
days (IQR)
40 days (25-85)
30 days (N/A)
39 days (32-43)
Abbreviations:
AHSCT- Autologous Haematopoietic Stem cell transplant; CT- computed tomography; DMT- Disease modifying therapy; EBV- Epstein Barr virus; EDSS- Kurtzke Expanded Disability Status Scale; IQR- Interquartile range; LPD- Lymphoproliferative disease; M-Protein: Monoclonal paraprotein or gammopathy; MS- Multiple Sclerosis
Table 1 Click here to access/download;Table;Tables 1-MS EBV .docx
Figure 1 Click here to access/download;Figure (.tif and .eps filesonly);Figure 1.1 EBV MS tif.tiff
Figure 2 Click here to access/download;Figure (.tif and .eps filesonly);Figure 2.1 EBV MS tif.tiff
Figure 3 Click here to access/download;Figure (.tif and .eps filesonly);Figure 3.1 EBV MS tif.tiff
EBV and Monoclonal Gammopathy of Clinical Significance in Autologous
Stem Cell Transplantation for Multiple Sclerosis.
Running Title: EBV complications in Auto-HSCT for MS
SUPPLEMENTARY:
Case vignettes of 2 MS patients describing EBV-related significant paraproteinaemia
and neurological sequelae.
Patient 1
43-year-old female with Relapsing Remitting Multiple Sclerosis (RRMS), previously treated
with natalizumab and three courses of alemtuzumab but continued to have breakthrough
disease. She had a relatively mild baseline disability with an Expanded Disability Severity
Scale (EDSS) of 2.5. She had an uncomplicated inpatient stay for the Autologous
Haematopoeitic Stem cell transplant (AHSCT) procedure and was discharged on day 15 post-
transplant. A blood test on day 26 demonstrated Epstein-Barr Virus (EBV) reactivation
(155,845 copies/ml). A repeat test on day 34 showed an increase in copy number to 638,634
copies/ml. She was asymptomatic, and the plan was to monitor this closely. On day 37 post-
transplant she developed a significant deterioration in strength in the right lower limb and on
day 42 she developed pyrexia and was admitted to a local hospital. She was found to have
CMV reactivation which was treated with IV ganciclovir as well as ongoing EBV reactivation
and she remained an inpatient for 4 weeks. She did not receive rituximab at the local centre
but on repeat testing at day 145 the copy number was vastly reduced at 2,355 copies/ml. A
high IgM paraproteinaemia was first detected at day 92 post-transplant (48.58g/L). This had
not been routinely monitored previously. This paraproteinaemia was initially felt to be
asymptomatic and was monitored closely, slowly improving over time. A CT scan was
performed which demonstrated a single 1.7cm right hilar lymph node requiring observation.
Supplemental -Case Vignettes Click here to access/download;Supplemental data/Appendix -published online only;SUPPLEMENTARY-MS EBV updated
A bone marrow aspirate showed a small excess of plasma cells (5-9%) on aspirate with no
other significant findings.
The EBV reactivation initially settled at 6 months post-transplant. At one-year post transplant
she had a persistent IgM paraprotein (23g/L) and her right leg weakness had continued to
progress with her EDSS now at 5.0. There was also a mild recurrence of EBV (DNA at 1,335
copies/ml). It was considered that as the onset of the right leg weakness had coincided with
the high level of EBV reactivation and paraproteinaemia that these factors may have driven a
peripheral neuropathy. She was treated with rituximab 375mg/m2 weekly for 4 weeks at 19
months post-transplant following which EBV DNA again became undetectable and the
paraprotein reduced to 9g/L. Despite this, there was no improvement in strength of the right
leg. Nerve conduction studies subsequently confirmed an L5-S1 radiculopathy but without a
generalised polyneuropathy neuropathy. She has had no new or active demyelinating lesions
on MRI head and spine post-transplant that would account for these symptoms and the slowly
progressive nature of the weakness does not suggest an MS relapse. The cause of the
weakness is likely an atypical IgM paraprotein associated radiculo-neuropathy was strongly
suspected.
Patient 2
42-year-old male with Secondary Progressive Multiple Sclerosis (SPMS), previously treated
with interferon and copaxone which were discontinued due to side effects and ongoing
relapses, respectively. He was then treated with natalizumab for 2 years but continued to
progress and was offered HSCT. He had a moderate level of baseline disability with an EDSS
of 5.5 (walking at least 100m unaided). The transplant procedure was complicated by
neutropenic sepsis which was treated successfully, and he was discharged on day 13 post-
transplant with no new neurological symptoms. He was readmitted on day 17 post-transplant
with pyrexia and rigors. Blood cultures grew Stenotrophomonas maltophilia and he was
treated for line sepsis with appropriate antibiotics and fully recovered. An EBV viraemia of
58,324 copies/ml was detected for the first time on this admission. On day 22 he had
developed new urinary urgency, diplopia and significant deterioration in mobility. This was felt
to represent either a pseudorelapse driven by infection or a true relapse and an MRI was
performed which demonstrated no new demyelinating lesions and no other significant
pathology. A repeat EBV DNA assessment on day 28 demonstrated a significant rise in EBV
viraemia to >10 million copies/ml (log change).
His neurological symptoms persisted and on day 34 he began spiking temperatures again;
antibiotics were restarted but blood and urine cultures came back negative, but his EBV
viraemia had risen to over 39 million copies/ml. He continued to experience intermittent
pyrexia, which possibly was attributed to his EBV viraemia. No evidence of lymphadenopathy
was noted during this period. Due to significant neurological decline, he was consequently
commenced on rituximab 375mg/m2 weekly for 4 weeks on day 38 post-transplant. Testing on
day 51 demonstrated a reduction in EBV viraemia to DNA of 2.2 million copies/ml and on day
52, a significant IgG kappa paraproteinaemia (45.6 g/L) was identified. This had not been
routinely monitored previously. It was considered that this degree of paraproteinemia and
resulting hyperviscosity may have been a driver of his neurological symptoms. These values
continued to improve over time with further doses of rituximab and the EBV vireamia was
<100,000 copies/ml and the IgG kappa paraprotein down to 8.63 g/L by Day 87. However,
due to persistence of these markers as well as his ongoing neurological symptoms, he was
given a single plasma exchange on day 80 that was of minimal symptomatic benefit.
He had ongoing rehabilitation, including a short admission in a specialist neuro-rehabilitation
ward. neurorehabilitation unit. At one year review he still required bilateral support to walk,
putting his EDSS at 6.5. A repeat MRI at 12 months post-transplant was again stable with no
new demyelinating lesions. This patient demonstrated significant deterioration in his condition
post-transplant and although there may be an element of disease progression, we suspect
this was in large part driven by EBV viraemia and associated paraproteinaemia/hyperviscosity.
The EBV viraemia was undetectable at the last follow up, although there was ongoing
paraproteinaemia with an IgG kappa of 15 g/L.
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EBV and Monoclonal Gammopathy of Clinical Significance in Autologous 1
Stem Cell Transplantation for Multiple Sclerosis. 2
Running Title: EBV complications in Auto-HSCT for MS 3
Varun Mehra*, Elijah Rhone*, Stefani Widya, Mark Zuckerman, Victoria Potter, Kavita Raj, 4
Austin Kulasekararaj, Donal McLornan, Hugues de Lavallade, Nana Benson-Quarm, Christina 5
Lim, Sarah Ware, Malur Sudhanva, Omar Malik, Richard Nicholas, Paolo A Muraro, Judith 6
Marsh, Ghulam J Mufti, Eli Silber, Antonio Pagliuca and Majid A. Kazmi 7
8
*These authors contributed equally to this work as 1st Authors. 9
10
Key Points: 11
EBV reactivation is common post-transplant with ATG for multiple sclerosis (MS), 12
with significant lymphoproliferative & neurological sequelae associated with rising 13
M-protein. Serial monitoring of EBV & M-protein is recommended post-transplant, as 14
is pre-emptive anti-CD20 therapy with EBV DNA >500,000 copies/ml. 15
16
Corresponding authors: 17
1. Dr Varun Mehra; [email protected]; +442032995378 18
Alternate Corresponding Author: 19
2. Dr Majid Kazmi; [email protected] ; +442071882757 20
21
Key Words: 22
Multiple Sclerosis; Autologous Hematopoietic Stem Cell Transplantation, Epstein-23
Barr Virus Infection; Monoclonal Gammopathy; Post-transplant Lymphoproliferative 24
Disorder 25
26
Highlighted Revision Click here to access/download;Highlighted Revision;EBV inMS updated CID revision -tracked copy 29.12.18.docx
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Author Affiliations: 27
1. Dr Varun Mehra*; Department of Haematology, King’s College Hospital NHS Foundation 28 Trust, Denmark Hill, London, United Kingdom. 29
2. Dr Elijah Rhone*: Department of Neurology, King’s College Hospital NHS Foundation Trust, 30 Denmark Hill, London, United Kingdom 31
3. Stefani Widya: GKT School of Medical Education, Kings College London University, 32 London 33
4. Dr Mark Zuckerman: Department of Virology, King’s College Hospital NHS Foundation 34 Trust, Denmark Hill, London, United Kingdom. 35
5. Dr Victoria Potter: Department of Haematology, King’s College Hospital NHS Foundation 36 Trust, Denmark Hill, London, United Kingdom. 37
6. Dr Kavita Raj: Department of Haematology, King’s College Hospital NHS Foundation Trust, 38 Denmark Hill, London, United Kingdom AND Department of Haematology, Guy’s and St. 39 Thomas’ NHS Foundation Trust, London, United Kingdom 40
7. Dr Austin Kulasekararaj: Department of Haematology, King’s College Hospital NHS 41 Foundation Trust, Denmark Hill, London, United Kingdom. 42
8. Dr Donal McLornan: Department of Haematology, King’s College Hospital NHS Foundation 43 Trust, Denmark Hill, London, United Kingdom AND Department of Haematology, Guy’s and 44 St. Thomas’ NHS Foundation Trust, London, United Kingdom 45
9. Dr Hugues de Lavallade: Department of Haematology, King’s College Hospital NHS 46 Foundation Trust, Denmark Hill, London, United Kingdom. 47
10. Nana Benson-Quarm: Department of Haematology, King’s College Hospital NHS 48 Foundation Trust, Denmark Hill, London, United Kingdom. 49
11. Christina Lim: Department of Haematology, King’s College Hospital NHS Foundation Trust, 50 Denmark Hill, London, United Kingdom. 51
12. Sarah Ware: Department of Haematology, King’s College Hospital NHS Foundation Trust, 52 Denmark Hill, London, United Kingdom. 53
13. Dr Malur Sudhanva: Department of Virology, King’s College Hospital NHS Foundation 54 Trust, Denmark Hill, London, United Kingdom 55
14. Dr Omar Malik: Department of Neurology, Imperial College Healthcare, London, United 56 Kingdom 57
15. Dr Richard Nicholas: Department of Neurology, Imperial College Healthcare, London, 58 United Kingdom 59
16. Professor Paolo A. Muraro: Department of Neurology, Imperial College Healthcare, 60
London, United Kingdom AND Department of Neuroimmunology, Imperial College London, 61 London, United Kingdom 62
17. Professor Judith Marsh: Department of Haematology, King’s College Hospital NHS 63
Foundation Trust, Denmark Hill, London, United Kingdom 64 18. Professor
Ghulam J. Mufti: Department of Haematology, King’s College Hospital NHS 65
Foundation Trust, Denmark Hill, London, United Kingdom 66 19. Dr Eli Silber: Department of Neurology, King’s College Hospital NHS Foundation Trust, 67
Denmark Hill, London, United Kingdom 68 20. Professor
Antonio Pagliuca: Department of Haematology, King’s College Hospital NHS 69
Foundation Trust, Denmark Hill, London, United Kingdom 70 21. Dr Majid Kazmi: Department of Haematology, King’s College Hospital NHS Foundation 71
Trust, Denmark Hill, London, United Kingdom AND Department of Haematology, Guy’s and 72 St. Thomas’ NHS Foundation Trust, London, United Kingdom 73
74
75
76
77
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Conflict of interest: The authors declare no competing financial interests as below: 78
1. Varun Mehra- no competing financial interests 79
2. Elijah Rhone- no competing financial interests 80
3. Stefani Widya- no competing financial interests 81
4. Mark Zuckerman- no competing financial interests 82
5. Victoria Potter- no competing financial interests 83
6. Kavita Raj- no competing financial interests 84
7. Austin Kulasekararaj- no competing financial interests 85
8. Donal McLornan- no competing financial interests 86
9. Hugues de Lavallade- no competing financial interests 87
10. Nana Benson-Quarm- no competing financial interests 88
11. Christina Lim- no competing financial interests 89
12. Sarah Ware- no competing financial interests 90
13. Malur Sudhanva- no competing financial interests 91
14. Omar Malik- no competing financial interests 92
15. Richard Nicholas- no competing financial interests 93
16. Paolo A Muraro- no competing financial interests 94
17. Judith Marsh- no competing financial interests 95
18. Ghulam J Mufti- no competing financial interests 96
19. Eli Silber- no competing financial interests 97
20. Antonio Pagliuca- no competing financial interests 98
21. Majid A. Kazmi- no competing financial interests 99
100
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Abstract 111
Introduction 112
Autologous haematopoietic stem cell transplantation (AHSCT) with anti-thymocyte globulin 113
(ATG) conditioning as treatment of active multiple sclerosis (MS) is rapidly increasing across 114
Europe (EBMT registry data 2017). Clinically significant Epstein Barr virus reactivation (EBV-115
R) following AHSCT with ATG for severe autoimmune conditions is an under-recognised 116
complication relative to T-cell deplete transplants performed for haematological diseases. 117
This retrospective study reports EBV-R associated significant clinical sequelae in MS patients 118
undergoing AHSCT with rabbit ATG. 119
Methods 120
Retrospective data was analysed for 36 consecutive MS-AHSCT patients at Kings College 121
Hospital, London. All patients routinely underwent weekly EBV DNA PCR monitoring and 122
serum electrophoresis for monoclonal gammopathy (MG or M-protein). EBV-R with rising 123
EBV viral load, M-protein and associated clinical sequelae were captured from clinical 124
records. 125
Results 126
All patients had evidence of rising EBV DNA-emia, including 7 who were lost to long term 127
follow-up, with a number of them developing high EBV viral load & associated 128
lymphoproliferative disorder (LPD). Nearly 72% (n-18/29) developed de-novo MG, some with 129
significant neurological consequences with high M-protein and EBV-R. Six patients required 130
anti-CD20 therapy (rituximab) with complete resolution of EBV related symptoms. Receiver 131
operating characteristics (ROC) estimated a peak EBV viraemia of >500,000 DNA copies/ml 132
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correlated with high sensitivity (85.5%) & specificity (82.5%) (AUC-0.87; p-0.004) in 133
predicting EBV-R related significant clinical events. 134
Conclusion 135
Symptomatic EBV reactivation increases risk of neurological sequelae and LPD in MS-136
AHSCT. We recommend regular monitoring for EBV and serum electrophoresis for MG be 137
mandated in MS patients in the first 3 months post AHSCT 138
139
140
141
142
143
144
145
146
147
148
149
150
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INTRODUCTION: 151
Multiple sclerosis (MS) is a chronic autoimmune, inflammatory, demyelinating disease of the 152
central nervous system[1]-[2], with a relapsing-remitting (RRMS) presentation in the majority 153
of patients at diagnosis. Recovery from relapses may be complete or partial[3],[4]. After a 154
variable period of time, people with RRMS may develop a more progressive disability 155
accumulation with or without superimposed relapses; termed secondary progressive multiple 156
sclerosis (SPMS). A minority experience progressive disability from the onset of disease, 157
termed primary progressive multiple sclerosis (PPMS)[4]. A number of immunomodulatory 158
disease modifying therapies (DMTs) are currently licensed for treatment of RRMS with an 159
aim of reducing number of relapses and accrual of disability, although with variable 160
efficacy[5]. Since 1996, Autologous Hematopoietic Stem Cell Transplantation (AHSCT) has 161
been a novel approach for MS management, using immunoablation followed by 162
immunomodulation mechanisms, with evidence of significant suppression of inflammatory 163
activity and qualitative changes in the reconstituted immune system (immune reset theory)[6–164
8]. AHSCT appears most effective for MS patients with evidence of inflammatory activity on 165
MRI, younger age, a shorter disease duration, low to moderate disability levels (Expanded 166
Disability Status Scale [EDSS] <6 or up to 6.5 if recent progression) and failure of at least 1 167
highly active DMT (natalizumab or alemtuzumab) with no significant comorbidities[9–11]. 168
Recently reported preliminary results of randomised MIST study[12] found AHSCT to be 169
superior to standard disease modifying therapy (DMT) for RRMS with respect to both 170
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treatment failure and disability progression. 171
172
However, risk of subsequent rise in opportunistic infections following such 173
immunosuppressive therapies remain a potential concern[13]. MS patients undergoing 174
AHSCT have often been exposed to a number of immunomodulating DMTs; the addition of 175
immunosuppressive rabbit anti-thymocyte globulin (rATG) to their conditioning regimen may 176
confer a higher risk of viral reactivation in these patients. The number of AHSCTs performed 177
for MS is rising significantly in Europe[14] and as more centres perform AHSCT for this 178
indication, it is increasingly important to recognise the unique problems faced by these 179
patients post AHSCT. This retrospective study reports for the first time, EBV-R associated 180
neurological sequelae and lymphoproliferative disorder (LPD) in MS patients undergoing 181
rATG conditioned AHSCT in our centre. 182
183
METHODS 184
Patients and procedures 185
Data was collected retrospectively on 36 consecutive MS patients undergoing AHSCT 186
between February 2012 and February 2017 at Kings College Hospital, London. Peripheral 187
blood stem cells were collected following standard mobilisation strategy consisting of 188
cyclophosphamide 4g/m2 over 2 days and granulocyte colony-stimulating factor for 7 days. 189
Thirty-five (97%) patients were conditioned using standard protocol of cyclophosphamide 190
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(50mg/kg for 4 days) and rATG (2.5mg/kg/day for 3 days) for in-vivo lymphodepletion, 191
followed by stem cell infusion. One patient was conditioned with 192
carmustine/etoposide/cytarabine/melphalan regimen along with an equivalent dose of rATG 193
(BEAM-ATG) prior to stem cell infusion. The median CD34 stem cell dose returned was 7.17 194
x10^6/kg (range 4.0-17.1x10^6/kg). 195
196
Prior exposure to EBV was assessed by serological evidence (EBV viral capsid antigen; VCA 197
IgG). EBV DNA load monitoring was performed on whole blood samples by standardised 198
quantitative real-time polymerase chain reaction (RT-PCR) using Rotor-GeneTM (Qiagen) 199
assay of EBV BZLF1 DNA. This assay was, adapted from our published assay using 200
LightCycler (Roche)[15] and since been validated against the recently published WHO 201
standard, with our lab’s EBV DNA quantification of 10 copies/ml considered equivalent to 10 202
IU/ml DNA reported with the WHO reference method[16]. .EBV-R was defined as rising EBV 203
DNA load of >10 copies/millilitre (ml) detected on two consecutive tests based on our assay 204
sensitivity. Symptomatic EBV-R was captured by peak blood EBV DNA load, presence of B 205
symptoms (defined by presence of either unexplained weight loss, recurrent fever, night 206
sweats); which was in-turn defined by clinical, radiological and/or histological evidence based 207
on recent ECIL-6 guidelines[17]. In addition, significant ‘clinical events’ were also defined as 208
new & persistent organ dysfunction (e.g. neurological events) temporally associated with 209
rising EBV viraemia in MS patients. Serum protein electrophoresis was routinely tested 210
around 3 months post HSCT, as part of our institutional practice, with immunoglobulin 211
subclasses identified by immunofixation electrophoresis. Patient outcomes were assessed at 212
last follow up as of April 2017. 213
214
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Statistics 215
The database of transplants and outcomes was built in Microsoft Excel 2016 and statistical 216
analyses were performed using IBM SPSS Statistics version 24.0. Patient characteristics are 217
presented as medians (with inter-quartile ranges; IQR) for data with non-normal distribution. 218
Comparisons of baseline characteristics used Mann-Whitney U test, Fisher’s exact test, or 219
Chi-squared test for trend as appropriate. Receiver Operating characteristics (ROC) curve 220
was obtained correlating LPD and clonal gammopathy associated clinical events with rising 221
EBV viraemia (copies/ml). 222
223
RESULTS 224
Baseline characteristics are presented in Table 1. Most MS patients (88.9%) had RRMS 225
phenotype with median of 2 (range 0-3) DMTs prior to AHSCT. Twenty-two MS patients had 226
prior exposure to natalizumab and 7 were treated with Alemtuzumab (6 patients received 227
both). All 36 (100%) MS patients were serologically positive for EBV VCA IgG pre-treatment, 228
indicating prior EBV exposure and had detectable EBV DNA post-AHSCT. Seven MS 229
patients were lost to long-term follow up for EBV monitoring. The median time to first EBV 230
DNA detection post-transplant was 30 days (IQR 23-46 days). EBV DNA levels peaked at a 231
median of 32 days post-transplant (IQR 31-53 days). All MS patients had normal baseline 232
lymphocyte counts (median) pre-HSCT with a median time of 46 days (range 14-404 days) 233
to lymphocyte recovery (defined by total lymphocyte count >1.0x106/ml) following AHSCT 234
(See Figure 1). A high proportion (86%; n-25/29) of the MS patients in active follow-up 235
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recovered lymphocyte counts around D56 with a median lymphocyte count of 1.56 (106 236
cells/ml); Four patients remained lymphopenic at last follow up. 237
238
All patients were stratified into following 3 groups according to peak rise/burden of EBV DNA-239
aemia (copies/ml): <100,000 (<100k) copies/ml, 100,001-500,00 (100k-500k) copies/ml and 240
>500,000 (>500k) copies/ml to identify any specific thresholds for clinically significant events 241
related to rising EBV-R (Table 1). The majority of patients (76%) with rising EBV viral load 242
>100k copies/ml were routinely screened by computed tomographic (CT) scans to assess for 243
evidence of LPD, in line with our institutional policy. One third (34.5%) of patients developed 244
peak EBV viraemia of >500k copies/ml. Eight patients (27.6%) developed symptomatic EBV-245
R; defined as persistent fever, lymphadenopathy and/or B symptoms. Of these 8 patients, 246
only 1 (12.5%) had a peak EBV viraemia <100kDNA copies/ml with the remaining 7 (87.5%) 247
patients having a peak EBV viraemia of >500k copies/ml. Three patients with rising EBV 248
viraemia >500k copies/ml had findings consistent with probable LPD on CT imaging; 249
however, none had definitive histological diagnosis. Three MS patients had worsening 250
neurological symptoms concurrent with rising EBV viraemia >500k copies/ml and clonal 251
gammopathy, as described below. 252
253
Interestingly, we also observed frequent de novo monoclonal gammopathy (MG or M-protein) 254
in 18 MS patients (62.4%) following rising EBV viraemia; the majority (n-16) of whom 255
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developed IgG subtype and the remaining 2 developed IgA and IgM M-protein. Concerningly 256
two of these patients developed clinically significant M-Protein burden; one patient with IgG 257
Kappa M-protein of 45.6g/L developed hyper-viscosity and neurological symptoms mimicking 258
MS relapse, requiring plasma exchange. Another patient developed significant lumbosacral 259
radiculopathy with rising EBV-R and high IgM lambda M-protein (IgM 48.5g/L) (see 260
supplementary case vignettes). Figure 2 highlights the association of neurological 261
symptom onset following rising EBV viraemia (log copies), falling lymphocyte counts (x 262
106/ml) with significant rise in M-protein (gm/lt) levels post AHSCT. A third patient developed 263
painful lower limb paraesthesia following rising EBV viraemia >500k copies/ml, although did 264
not have any M-protein detected. Their symptoms persisted at last follow up despite no 265
evidence of MS related new disease activity. 266
267
Six patients were treated with anti-CD20 antibody, rituximab (375mg/m2 weekly up to 4 268
weeks), due to clinical severity of EBV reactivations and, leading to 1reduction in EBV viral 269
load and concurrent improvement/resolution of EBV related symptoms. ROC curve analysis 270
(Figure 3) confirmed EBV viraemia of >500k copies/ml correlated with high sensitivity 271
(85.5%) and specificity (82.5%) (AUC-0.87; 95%CI-0.73-1.0; p-0.004) in predicting significant 272
EBV related clinical events (evidence of LPD and/or neurological symptoms) that may require 273
treatment with rituximab. The sensitivity dropped significantly on lower estimates for events 274
below 500k copies/ml. 275
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276
The median time to resolution of EBV viraemia post-rituximab was 21 days (IQR 19-124 days) 277
in 5 patients with >500,000 k copies/ml (one patient was treated for late onset persistent 278
symptomatic clonal gammopathy, despite fall in EBV levels). No significant adverse events 279
were noted in the treated group. Nine patients had a persistent low level EBV viraemia 280
detectable at last follow up. All patients who underwent AHSCT were alive as of April 2017. 281
282
DISCUSSION: 283
MS as an autoimmune disorder (AD) has is theorised to have generally similar underlying 284
pathophysiological immune dysregulation mechanisms[18–22] relative to other chronic 285
autoimmune conditions. Epstein-Barr virus (EBV) is increasingly implicated in the 286
pathogenesis of MS by virtue of epidemiological and serological evidence, impaired CD8+ T 287
cell immune responses to EBV and possible underlying genetic susceptibility for 288
autoimmunity (with EBV encoded protein interactions), as recently described by Harley et al 289
and others[1,23–25]. 290
291
Generally, EBV-R related LPD has been reported in both allogeneic (allo-HSCT) and solid 292
organ transplants treated with immunosuppressive therapy, often with a significant impact 293
on organ function and overall survival[26–30]. It is observed that reduced intensity allo-HSCT 294
for malignant haematological conditions using alemtuzumab have a relatively lower overall 295
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risk of LPD compared to ATG based treatments, possibly mediated by more effective pan-B 296
& T cell lymphodepletion. In contrast, ATG primarily affects the T-cell repertoire with delayed 297
EBV specific CD8+ T cell recovery[31]. Clinically significant endogenous viral infections 298
including EBV following ATG conditioned AHSCT for severe ADs such as Crohn’s disease 299
and sSystemic sSclerosis is increasingly recognised, but the development of 300
lymphoproliferative disorders (LPD) remains rare in these ADs[13,32,33]. Nash et al[32] 301
concerningly reported 2 deaths (1 MS and 1 systemic sclerosis patient) from EBV related 302
LPD in 56 ATG conditioned AHSCT for autoimmune diseases. Additionally, EBV associated 303
haemophagocytosis in ATG-AHSCT for ADs have also been reported[34], with one resulting 304
in death of the patient[35]. 305
306
Our report of suspected EBV related LPDs (~10%) in MS-AHSCT group is relatively higher 307
than published reports with allo-HSCTs (4.5-7%)[36,37] and our own centre’s unpublished T-308
cell depleted allo-HSCT experience (6.5%); possibly a reflection of underlying 309
immunopathological state of MS itself[38]. This is further corroborated by the fact that similar 310
LPD risk has not been observed in other ADds, e.g. Crohn’s disease,, treated with ATG -311
AHSCT in this centre. Another example from ouour centre’s experience of in severe aplastic 312
anaemiaa (n-40), a type of AD causing severe bone marrow failure(n-40) treated & treated 313
with ATG/ciclosporin,n; only 52% (n-21/40) patients developed EBV-R (unpublished data) 314
and . nNone had LPD or required any treatment, supporting the notion that it may not just be 315
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a specific ATG related problem.suggesting that the problem may not be ATG specific. 316
317
Our study’s observation of significant persistent neurological events (with no evidence of new 318
MS disease activity) associated with clonal gammopathy, suggest a potentially new clinical 319
syndrome, described for the first time in ATG conditioned AHSCTs in MS and, possibly 320
induced by clonal B cell dysregulation following EBV-R. It could be hypothesised that any 321
remaining EBV infected latent B cells, which may still have survivedsurviving despite high 322
doses of cyclophosphamide (given with mobilisation and conditioning in MS ASHCT)[8] and 323
compounded by depletion of CD8+ T cells by ATG, may serve as potential source for EBV 324
escape while interacting abnormally within the host immune micro-environment[39] and 325
leading to rise in M-protein, LPD and neuro-inflammatory insults in some of the MS patients 326
post AHSCT. Reports of lower incidence of EBV-R and LPD in another commonly used 327
protocol for MS-AHSCT using BEAM-ATG (Ricardo Saccardi, Carregi University Hospital, 328
Florence; personal communication) may reflect the greater myeloablative effect of BEAM 329
chemotherapy which could further deplete the residual B cell pool and thus lower potential 330
for EBV proliferation. It is plausible that dose of rATG is critical, given we have not seen 331
similar reports from other centres where less rATG doses were given for MS-AHSCT (range 332
between 5.0-6.5 mg/Kg; personal communication), but there seems to be some variability in 333
prospective serial EBV monitoring in these patients. 334
335
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The clinical threshold for EBV viral load as a significant risk factor for post-transplant LPD is 336
widely debated. This study reports peak EBV viral load >500k copies/ml post AHSCT is 337
significantly associated with probable LPD and neurological events in MS patients with high 338
sensitivity (85.5%) and specificity (82.5%) (p-0.004) (Fig 3, ROC curve). Our ROC curve 339
estimates are potentially limited by the relatively small number of events analysed but this 340
has consistently been useful in our MS-AHSCT experience for predicting clinical events with 341
high EBV load. Our EBV PCR assay has been validated against the recently defined standard 342
WHO reference method (i.e. 10 copiesy/ml=10 IU/ml EBV DNA) [16] and thus this EBV 343
threshold for pre-emptive treatment with Rituximab, can potentially be applied in relevant 344
clinical context in other centres using similar validated essays. Rituximab treatment delivered 345
good overall response in our symptomatic patients, with resolution of EBV related clinical 346
symptoms and no subsequent viral or bacterial infections at last follow up. The role of 347
prophylactic or pre-transplant rituximab in MS-AHSCT is also a potential area of interest in 348
reducing risk of EBV-LPD in stem cell transplants as observed by Burns et al[36] and future 349
randomised studies are required to investigate its potential benefit. 350
351
Our study limitations include its retrospective nature and that no suspected LPD patients had 352
histological confirmation, mainly related to patient refusal or technical difficulties. Seven MS 353
patients were lost to follow up for EBV monitoring following discharge, which limits the 354
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findings of this study. Additionally, our numbers were too small to identify any association of 355
EBV related clinical events with previous DMT exposure in MS patients. 356
357
In conclusion, symptomatic EBV reactivation increases risk of neurological sequelae and 358
LPD in MS-AHSCT. Regular monitoring for rising EBV viraemia, as recommended by 359
Snowden et al [10] and Muraro et al [11], and serum electrophoresis for M-protein should be 360
considered mandated in the first 3 months post-AHSCT for MS. and Wwe recommend 361
persistent high EBV viraemia > 500k DNA copies/ml as potential trigger for consideration of 362
pre-emptive anti-CD20 therapy and potentially reduce associated morbidity. 363
364
365
366
367
368
369
Acknowledgements: To our patients and their families and carers in supporting this study. 370
371
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374
375
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507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
- 21 -
Table 1: Baseline patient characteristics and EBV related clinical events according to peak 527
EBV DNA-aemia burden. 528
529
Baseline characteristics (n-36)
Patient Groups according to peak
EBV DNA in copies/ml
(n-29)
0 - 100,000 100,001
- 500,000
>500,000
Median age at time of AHSCT in years (range)
43.5 (36– 47)
No of patients (%) 16 (55.2) 3 (10.3) 10 (34.5)
Gender Male Female
19 (52.8%) 17 (47.2%)
M-Protein (n) 11 0 7
Disease Type (n; %) Relapsing Remitting MS Secondary Progressive MS Primary Progressive MS
22 (61.1%) 10 (27.8%) 4 (11.1%)
Median EBV DNA log value at peak
(IQR)
4.8 (3.5-4.8)
5.5 (N/A)
6.25 (6.1-6.9)
Median number of previous DMT (range)
2 (0 – 6) Median number of
prior DMTs 2 3 2
Previous use of high efficacy DMT (n) Natalizumab Alemtuzumab Both
22 8 6
Symptomatic EBV (n)
1 0 7
Median EDSS (range) 6.0
(2.5 – 8.0) LPD diagnosis (CT/Biopsy) (n)
0 0 3
by CT alone
Median follow up post AHSCT in days (range)
436 (188 – 785)
Neuro/autoimmune complications (n)
0 0 3
No. of patients with prior EBV exposure (n; %)
36 (100%) Treated with Rituximab (n)
0 0 6
No. of patients with detectable EBV post AHSCT (n; %)
29 (80.5%) Confirmed EBV resolution at last
follow up (n) 7 2 7
No. of patients lost to long term follow up (n; %)
7 (19.5%) Detectable EBV DNA at last follow up (n)
9 1 3
Median Time to EBV detection post AHSCT in days (IQR)
30 (23-46) Median time for EBV
resolution (IQR in days)
67 days (44-155)
47 days (N/A)
63 days (45 - 170)
Median Time to peak EBV DNA levels in days (IQR)
32 (31-53) Median Time to peak EBV DNA levels in
days (IQR)
40 days (25-85)
30 days (N/A)
39 days (32-43)
530
Abbreviations: 531
AHSCT- Autologous Haematopoietic Stem cell transplant; CT- computed tomography; DMT- 532
Disease modifying therapy; EBV- Epstein Barr virus; EDSS- Kurtzke Expanded Disability 533
Status Scale; IQR- Interquartile range; LPD- Lymphoproliferative disease; M-Protein: 534
Monoclonal paraprotein or gammopathy; MS- Multiple Sclerosis 535
- 22 -
Figure Legends 536
537
Figure 1: Trend of Lymphocyte count recovery following ATG in MS patients. 538
539
Legend: This figure shows trends of lymphocyte count from baseline to recovery post 540
AHSCT for MS patients. Majority of patients had normal baseline lymphocyte counts pre-541
HSCT and became lymphopenic post ATG with counts recovering towards d+28 and >85% 542
of patients recovered counts by d+56, with some overshooting from their baseline, possibly 543
reflective of EBV related lymphoproliferation in some of these patients. 544
545
AHSCT: Autologous Haematopoietic Stem Cell Transplantation; ATG: Anti-Thymocyte 546
Globulin; d+: Days post AHSCT; MS: Multiple Sclerosis. 547
548
549
Figure 2: Paraprotein, EBV & Lymphocyte trends in two MS patients with significant 550
neurological sequelae post AHSCT. 551
552
Legend: This figure demonstrates trends of EBV copies (log), paraprotein levels (g/lt) and 553
Lymphocyte levels (counts x10^6/ml) in two MS patients with significant neurological 554
symptoms following EBV reactivation. Both patients had significant EBV viraemia (log>5.2 555
or >500,000 copy number) and developed significant paraproteinaemia, which was only 556
noted after persistent unexplained neurological symptoms. The trend reversed following 557
anti-CD20 (rituximab) therapy, with limited recovery in neurological symptoms. 558
559
D: Days post AHSCT; EBV-R: Epstein Barr Virus reactivation; MS: Multiple sclerosis; 560
AHSCT: Autologous Haematopoietic Stem Cell Transplants. 561
562
563
Figure 3: ROC curve estimates for peak EBV viraemia levels and significant clinical 564
events in MS post AHSCT. 565
566
Legend: ROC curve demonstrating significant correlation between high EBV levels and 567
clinical events (LPD & neurological events) in MS-AHSCT patients, with highest sensitivity 568
and specificity noted with peak EBV viraemia of >500,000 copies/ml (p-0.0004). 569
570
EBV: Epstein Barr Virus; LPD- Lymphoproliferative disorder; MS-AHSCT: Multiple Sclerosis 571
patients treated with autologous haematopoietic stem cell transplants; ROC: receiver 572
operating characteristics 573
574 575 576 577 578 579 580
581
- 23 -
582
Figure 1: Trend of Lymphocyte count recovery following ATG conditioned AHSCT in 583
MS patients. 584
585
586 587
588
Abbreviations: 589
AHSCT: Autologous Haematopoietic Stem Cell Transplantation; ATG: Anti-Thymocyte 590
Globulin; d+: Days post AHSCT; MS: Multiple Sclerosis. 591
592
593
594
595
596
597
598
599
Lymphocyte count trends post ATG in MS-AHSCT
Time (in days) since ATG in MS-AHSCT
- 24 -
Figure 2. Paraprotein, EBV & Lymphocyte trends in two MS patients with significant 600
neurological sequelae post AHSCT 601
602
603 604 605 606 607 608
609
Abbreviations: 610
D: Days post AHSCT; EBV-R: Epstein Barr Virus reactivation; MS: Multiple sclerosis; 611
AHSCT: Autologous Haematopoietic Stem Cell Transplants. 612
613
614
615
616
617
618
619
620
621
- 25 -
622
Figure 3: ROC curve estimates for peak EBV viraemia levels and significant clinical 623
events in MS post AHSCT. 624
625
626
627
Abbreviations: 628
629
EBV: Epstein Barr Virus; LPD- Lymphoproliferative disorder; MS-AHSCT: Multiple Sclerosis 630
patients treated with autologous haematopoietic stem cell transplants; ROC: receiver 631
operating characteristics 632
633
634
635
636
637
638
639
640
EBV DNA in
copies/ml
Sensitivity Specificity
0-50,000 100% 50%
50,001-
100,000
85.5% 60.1%
100,001-
500,000
85.5% 73.7%
>500,001 85.5% 82%
>700,000 71.4% 82%
AUC-0.87 (95% CI: 0.73-1.0) AUC-0.87 (95% CI: 0.73-1.0); p-0.004
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